The effect of euthanasia technique on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility

This study was designed to determine the effects that specific euthanasia methods have on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility. Rats were euthanatized by decapitation (DC), pentobarbital overdose (PB), or anesthesia with CO2, methoxyflurane or ether followed by DC (CO2-DC, Met-DC, Ether-DC, respectively). Rabbits were killed by a similar protocol, but CO2 overexposure replaced Ether-DC. The rat and rabbit aortas produced mainly 6-keto PGF1 alpha, the prostacyclin metabolite, and lesser amounts of PGE2. No qualitative differences were seen in arachidonate metabolites. However, aortic tissue from rabbits and rats killed by Met-DC produced more prostacyclin. In contrast, aorta from rabbits euthanatized by CO2-DC produced less prostacyclin than controls, whereas aorta from rats killed in the same way yielded greater amounts of prostacyclin. Aortic tissue from rabbits killed by Met-DC and CO2-OD was less responsive to acetylcholine (ACH). Intestinal contractility to ACH was increased in rabbits when Met-DC was used as the method of euthanasia, while colon from rats sacrificed by Met-DC showed decreased responsiveness to ACH. Colon from rats killed by intraperitoneal PB exhibited altered contractility to ACH and norepinephrine. The results of this study show that methoxyflurane, carbon dioxide (rabbit) and pentobarbital (rat) alter the vascular synthesis of prostacyclin and smooth muscle contractility. We conclude that the method of euthanasia affects certain physiologic parameters and careful consideration should be given to the selection of a particular euthanasia technique.     

Intracellular calcium buffering capacity in isolated squid axons

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 μmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/μM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 μM is taken up by this system.     

The binding mechanism,multiple binding modes,and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

Sortase enzymes are vitally important for the virulence of gram‐positive bacteria as they play a key role in the attachment of surface proteins to the cell wall. These enzymes recognize a specific sorting sequence in proteins destined to be displayed on the surface of the bacteria and catalyze the transpeptidation reaction that links it to a cell wall precursor molecule. Because of their role in establishing pathogenicity, and in light of the recent rise of antibiotic‐resistant bacterial strains, sortase enzymes are novel drug targets. Here, we present a study of the prototypical sortase protein Staphylococcus aureus Sortase A (SrtA). Both conventional and accelerated molecular dynamics simulations of S. aureus SrtA in its apo state and when bound to an LPATG sorting signal (SS) were performed. Results support a binding mechanism that may be characterized as conformational selection followed by induced fit. Additionally, the SS was found to adopt multiple metastable states, thus resolving discrepancies between binding conformations in previously reported experimental structures. Finally, correlation analysis reveals that the SS actively affects allosteric pathways throughout the protein that connect the first and the second substrate binding sites, which are proposed to be located on opposing faces of the protein. Overall, these calculations shed new light on the role of dynamics in the binding mechanism and function of sortase enzymes.     

Peripheral nuclear matrix actin forms perinuclear shells

Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers. J. Cell. Biochem. 70:240–251, 1998. © 1998 Wiley-Liss, Inc.     

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

Background  

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear.     

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.     

Solid-phase microextraction and the human fecal VOC metabolome

The diagnostic potential and health implications of volatile organic compounds (VOCs) present in human feces has begun to receive considerable attention. Headspace solid-phase microextraction (SPME) has greatly facilitated the isolation and analysis of VOCs from human feces. Pioneering human fecal VOC metabolomic investigations have utilized a single SPME fiber type for analyte extraction and analysis. However, we hypothesized that the multifarious nature of metabolites present in human feces dictates the use of several diverse SPME fiber coatings for more comprehensive metabolomic coverage. We report here an evaluation of eight different commercially available SPME fibers, in combination with both GC-MS and GC-FID, and identify the 50/30 μm CAR-DVB-PDMS, 85 μm CAR-PDMS, 65 μm DVB-PDMS, 7 μm PDMS, and 60 μm PEG SPME fibers as a minimal set of fibers appropriate for human fecal VOC metabolomics, collectively isolating approximately 90% of the total metabolites obtained when using all eight fibers. We also evaluate the effect of extraction duration on metabolite isolation and illustrate that ex vivo enteric microbial fermentation has no effect on metabolite composition during prolonged extractions if the SPME is performed as described herein.     

Molecular population genetics of the alcohol dehydrogenase locus in the Hawaiian drosophilid D. mimica

Sequence variation among 10 alleles of the alcohol dehydrogenase (Adh) gene of the Hawaiian drosophilid D. mimica was analyzed with reference to the evolutionary history of the Hawaiian subgroup as well as to levels and patterns of polymorphism of the Adh gene in continental drosophilid species. The Adh gene of D. mimica is less polymorphic than that of other drosophilid species, and no replacement substitutions were found. Statistical analyses of the Adh alleles suggested the action of balancing selection and revealed significant linkage disequilibrium among three of the variable sites. The effective population size was estimated to be only slightly smaller than that of continental species and, surprisingly, on the same order of magnitude as the actual size.